EP0829488A1 - Antigene peptidsubstanz und immotestverfahren - Google Patents

Antigene peptidsubstanz und immotestverfahren Download PDF

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EP0829488A1
EP0829488A1 EP95917489A EP95917489A EP0829488A1 EP 0829488 A1 EP0829488 A1 EP 0829488A1 EP 95917489 A EP95917489 A EP 95917489A EP 95917489 A EP95917489 A EP 95917489A EP 0829488 A1 EP0829488 A1 EP 0829488A1
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arg
hcv
pro
ser
lys
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French (fr)
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EP0829488A4 (de
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Toshiaki Hachioji Laboratories KUMAZAWA
Yoshiyasu Hachioji Laboratories KIYA
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SRL Inc
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SRL Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/18—Togaviridae; Flaviviridae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to an antigenic peptide compound and an immunoassay which are useful for measuring an antibody relating to HCV (hepatitis C virus).
  • viruses exist on the earth, and some of them are pathogens.
  • a hepatitis virus as a cause of viral hepatitis is transmissible at the time of blood transfusion, injection, childbirth, etc.
  • the blood transfusion is one of the important measures for maintaining the life of a human being.
  • the blood transfusion causes viral hepatitis.
  • the viral hepatitis is classified into hepatitis A, hepatitis B and non-A non-B hepatitis.
  • HBV hepatitis B virus
  • a diagnostic reagent for detecting the presence of the HBV has been developed, and it has become possible to easily confirm the presence of the HBV, prior to the blood transfusion. Accordingly, the HBV infection of a human being due to the blood transfusion has entirely been prevented.
  • non-A non-B hepatitis hepatitis A virus
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • the above-mentioned paper describes the following experiments. That is, a preparation of blood coagulation Factor VIII which was the same as the preparation which had caused the non-A non-B hepatitis when administered to a patient, was administered to a Chimpanzee-No. 1 thereby to cause non-A non-B hepatitis. Further, a preparation extracted from the liver of the above Chimpanzee-No. 1 was similarly administered to a Chimpanzee-No. 2 thereby to cause non-A non-B hepatitis, and thereafter, the blood plasma of this Chimpanzee-No. 2 was extracted. The resultant blood plasma of the Chimpanzee-No. 2 was further administered to a Chimpanzee-No.
  • Type-II Journal of Virology, 65(3), 1105 - 1113, 1991
  • Type-III Journal of General Virology, 72, 2697 - 2704, 1991
  • Type-IV Virology, 188, 331 - 341, 1992.
  • the administration of the interferon produces a strong side effect such as alopecia and fever, and the load thereof to the patient is heavy. Therefore, the determination of the HCV genotype prior to the treatment is extremely important in view of the prediction of the effect of the interferon administration.
  • the HCV RNA is extracted from the specimen of a patient, a cDNA complementary to the resultant RNA is synthesized, the cDNA is amplified by a PCR (polymerase chain reaction) method, and a band corresponding to the thus amplified cDNA is identified by use of electrophoresis, thereby to determine the genotype. Since such a procedure includes complicated steps and usually lasts for 48 hours or more, it requires a long period of time and a high cost.
  • HCV antibodies in the blood sera of patients can be classified into two types by using an antigen (including about 300 amino acids) which has been prepared by a genetic recombination technique (KAN TAN SUI, 22, 883-889, 1991).
  • an antigen including about 300 amino acids
  • KAN TAN SUI, 22, 883-889, 1991 a genetic recombination technique
  • the administration of the interferon requires a high cost
  • the determination of the above-mentioned genotype which is a prerequisite for the interferon administration
  • has a disadvantage such that the determination per se also requires a high cost.
  • the genotype of the HCV can be determined easily prior to the interferon administration, it becomes possible to easily predict the effect of the interferon administration so as to properly design the method of administrating the interferon and a guideline for the treatment using the same.
  • an object of the present invention is to provide an antigen for measuring an HCV antibody, which enables simple determination of the HCV antibody wherein the genotype-I and genotype-II can be distinguished from the genotype-III and genotype-IV.
  • Another object of the present invention is to provide an antigen for measuring an HCV antibody, which enables the determination of the HCV antibody wherein the genotype-I and genotype-II can be distinguished from the genotype-III and genotype-IV; and further a cross-reaction or non-specific reaction is suppressed.
  • a further object of the present invention is to provide a method of simply measuring an HCV antibody, wherein the genotype-I and genotype-II can be distinguished from the genotype-III and genotype-IV.
  • a further object of the present invention is to provide a method of simply measuring an HCV antibody, wherein the genotype-I and genotype-II can be distinguished from the genotype-III and genotype-IV; and further a cross-reaction or non-specific reaction is suppressed.
  • a further object of the present invention is to provide a method of measuring an HCV antibody at a low cost, wherein the genotype-I and genotype-II can be distinguished from the genotype-III and genotype-IV.
  • an HCV antibody type serotype
  • a specific amino acid sequence based on the an HCV antigenic site a sequence comprising at least six amino acids
  • the antigenic peptide compound according to the present invention is based on the above discovery, and more specifically, it comprises: a sequence which comprises at least six sequential amino acids and is included in an amino acid sequence represented by the following formula (1): Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Ala-Arg-Arg-Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro ⁇ (1)
  • the present invention also provides an antigenic peptide compound, comprising a sequence which comprises at least six sequential amino acids and is included in an amino acid sequence represented by the following formula (2): Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Asp-Arg-Arg-Ser-Thr-Gly-Lys-Ser-Trp-Gly-Lys-Pro ⁇ (2)
  • the present invention further provides an immunological method of measuring an HCV antibody, comprising: fixing any of the antigenic peptides represented by the above formula (1) and/or (2) to a carrier; binding an HCV antibody in a specimen to the peptide by utilizing an antigen-antibody reaction; and binding a labeled ligand to the HCV antibody by utilizing an antigen-antibody reaction, thereby to measure the HCV antibody in the specimen.
  • ligand refers to a substance (such as antibody and/or antigen) having a specific binding affinity to an antigen and/or antibody.
  • the type of HCV is determined by utilizing the sequence of the HCV gene per se.
  • the type of HCV is determined by utilizing the type of an antibody (serotype) corresponding to an antigenic site peculiar to the HCV.
  • Such determination of the antibody type according to the present invention may be conducted within a much shorter period of time as compared with the period of time required for the conventional genotype determination (extraction of HCV RNA ⁇ synthesis of cDNA ⁇ amplification of the cDNA by PCR method ⁇ identification of the amplified band using electrophoresis).
  • the operation steps constituting the antibody type determination according to the present invention are simple. As a result, according to the present invention, there may be provided a method of simply determining the HCV type (genotype) at a low cost.
  • the cross reaction or non-specific reaction which can occur along with an antigen-antibody reaction is effectively suppressed, as compared with that in the case of the conventional method of determining the serum type by utilizing an antigen comprising about 300 amino acids.
  • the sensitivity to the core region is increased according to the present invention, for example, from an epidemiological viewpoint, it is possible to distinguish the past infection of HCV (e.g., based on the determination of the core region as a virus main body) and the current infection thereof (e.g., based on the determination of NS4 and/or NS5 region appearing at the time of the virus propagation) by using the serotype.
  • the peptide appearing at the time of the virus propagation such as NS4 and NS5 regions immediately disappears after the cure of the hepatitis, but on the other hand, the core region can be detected even within a predetermined period of time after the cure of the hepatitis (e.g., about one to two years after the cure). Therefore, according to the present invention, it is also possible to distinguish the past HCV infection on the basis of the detection of the core region.
  • Figs. 1A to 1C are graphs showing the results which have been obtained by calculating the "Hydrophilicity Value", “Hydropathy Value” and “Antigenicity Value” in Example 1 with respect to the core region of HCV appearing hereinafter.
  • Fig. 2 is a chromatogram showing the results of HPLC analysis of an antigenic peptide compound (1) (ckk-c12) obtained in Example 2.
  • Fig. 3 is a chromatogram showing the results of HPLC analysis of an antigenic peptide compound (2) (ckk-c12) obtained in Example 2.
  • Fig. 4 is a view schematically showing the relationships among the HCV antigenic regions and the respective antigenic peptides.
  • Fig. 5 (Table 1) is a table showing the results of HCV serotype determination which have been obtained in Examples appearing hereinafter.
  • amino acid sequences in the present specification the respective amino acids constituting an amino acid sequence are described in a direction of from the N-terminal amino acid (left end) toward the C-terminal amino acid (right end).
  • amino acid sequence (1) “Arg” is the N-terminal
  • Pro is the C-terminal.
  • amino acid descriptions are used.
  • the antigenic peptide compound according to the present invention is a peptide compound which has a sequence included in the following formula (1) or (2) having thirty (30) amino acids, and includes a sequence of at least 6 (six) sequential amino acids.
  • any of sequences which have various lengths constitute the sequence represented by the above formula (1) or (2), and include at least sequential six amino acids may preferably be used as an antigen, similarly as the peptide (1) and/or (2).
  • the number of the amino acids constituting each of the "amino acid sequences having various lengths” may preferably be 21 or larger (more preferably 25 or larger), in view of the balance between the ability to discriminate HCV, and the suppression of the cross-reaction/non-specific reaction.
  • amino acid sequences having various lengths may include those as shown below.
  • any of peptides which comprise the sequence represented by the above formula (1) or (2) may preferably be used as an antigen, similarly as the peptide (1) or (2).
  • the number of the amino acids constituting such a peptide may preferably be 40 or less when the peptide is chemically synthesized; and may preferably be 100 or less when the peptide is synthesized by using a recombinant technique.
  • the method of obtaining the above-mentioned antigenic peptides is not particularly limited.
  • the peptide may be synthesized by using a chemical method, or may be produced by using a genetic engineering technique (for example, genetic recombinant technique, i.e., recombinant method).
  • a chemical technique it is preferred to link the respective amino acids by use of a solid-phase technique, in view of easiness in the purification of an intermediate product, etc.
  • it is further preferred to synthesize the peptide by means of an automatic peptide synthesizing apparatus (e.g., "430 A" Peptide Synthesizer mfd. by Applied Bio-Systems Co.).
  • the sequences of the above peptides (1) and (2) have been discovered on the basis of the "extraction" of an antigenic site from the amino acid sequence of the core region of HCV (as described in Example 1 appearing hereinafter). Further, the present inventor has investigated the HCV genotype by use of an enzyme immunoassay technique (ELISA or EIA) using these peptides (1) and (2), and has confirmed the specificity of such a technique by using specimens of patients, the HCV genotypes of which have separately been identified (as described in Examples 4 and 5 appearing hereinafter).
  • ELISA or EIA enzyme immunoassay technique
  • any of sequences which have various lengths constitute the sequence represented by the above formulas (3) to (6) comprising 40 or 20 amino acids, and includes at least six sequential amino acids may preferably be used as an antigen, similarly as each of the peptides (3), (4), (5), and/or (6).
  • amino acid sequences having various lengths may include those as shown below.
  • any of peptides (including amino acids of 16 - 31 or more) which comprise any of the sequences represented by the above formulas (3) to (6) may preferably be used as an antigen, similarly as each of the peptides (3) to (6).
  • the number of amino acids constituting such a peptide may preferably be 40 or less when the peptide is chemically synthesized; and may preferably be 100 or less when the peptide is synthesized by using a recombinant technique.
  • the order of preference in the use thereof as an antigen is as follows.
  • the peptides (1) and/or (2) are inclusively referred to as "core”
  • the peptides (3) and/or (4) are inclusively referred to as "NS-4"
  • the peptides (5) and/or (6) are inclusively referred to as "NS-5”.
  • the order of preference in the use thereof as an antigen is as follows, in view of the accuracy in the serotype determination.
  • the HCV antibody in a sample or specimen may be measured immunologically.
  • the immunological measuring method to be used in the present invention is not particularly limited, but it is possible to utilize any of known immunoassay methods without particular limitation.
  • Specific examples of such immunoassay methods may include: enzyme immunoassay, radio immunoassay (RIA) fluorescence immunoassay (FIA), etc.
  • RIA radio immunoassay
  • FFA fluorescence immunoassay
  • an HCV antibody may be immunologically measured by use of the above-mentioned antigenic peptide, whereby the kind (serotype) of the HCV antibody contained in a specimen may be determined (For example, as described hereinbelow, it is possible to determine whether the serotype of the HCV antibody is classified into group-I or group-II).
  • group-I of HCV serotype corresponds to the HCV genotype-I and -II
  • the group-II of HCV serotype corresponds to the HCV genotype-III and -IV. Accordingly, it is possible to easily determine the HCV genotype.
  • the antigenic peptide is attached or solid phase-fixed to a carrier (or support), and the kind of the antibody against the HCV antigen (i.e., serotype of HCV) is determined by utilizing the specific affinity of the antigenic peptide to the HCV antibody.
  • a carrier or support to be used for such a purpose may include: bovine serum albumin (BSA), a polypeptide preferably having a molecular weight of about 5 ⁇ 10 4 to 10 ⁇ 10 4, wells of a micro-plate, a polystyrene ball (or polystyrene bead) preferably having a diameter of about 0.1 ⁇ m to 6 mm, etc.
  • the above peptides (1) to (2) may suitably be used for the measurement of an HCV antibody, and may also be used for an antigen or immunogen for producing a vaccine for HCV.
  • a vaccine may be prepared by using a genetic engineering technique (recombinant technique).
  • the antigenic site thereof was evaluated by using a method described in Hopp TP; Proc. Natl. Acad. Sci. 78, 3824 - 3828 (1981). More specifically, the evaluation was conducted in the following manner by using the above "Hydrophilicity Value" of amino acids.
  • the Hydrophilicity Values of respective sequential six amino acids were summed up. Such calculations were conducted with respect to all the regions of the amino acid sequences of HCV, thereby to calculate the strength of their hydrophilic properties and hydrophobic properties. The region showing a high hydrophilic property was treated as a site showing an antigenicity.
  • regions capable of being an antigen of hepatitis C were extracted. Further, among the thus determined regions, regions having a common amino acid sequence with respect to the genotype-I and -II, and regions having a common amino acid sequence with respect to the genotype-III and -IV were determined. As a result of such evaluation, there were discovered the following ckk-c12 and ckk-c22 as antigenic amino acid sequences for the core region satisfying such conditions.
  • HCV antigenic peptides (ckk-c12) and (ckk-c22) were synthesized in the following manner.
  • the peptides were synthesized by means of an automatic peptide synthesizing apparatus of "430A" Peptide Synthesizer mfd. by Applied Biosystems, by use of symmetric anhydrides of t-Boc amino acids as reagents.
  • the resultant synthesized peptide was dissolved in anisole dimethyl sulfide para-thio cresol, and thereafter, was subjected to a reaction at 0 - 5 °C for one hour in the presence of hydrofluoric acid, thereby to remove the protecting group (with reference to S. Sakakibara, Bull. Chem. Soc. Jpn., 40, 2164, (1967)).
  • the resultant crude crystals obtained by the removal of the protecting group was dissolved in 2N-acetic acid, and then extracted by use of ether, and the resultant extract was freeze-dried.
  • the freeze-dried product was purified by means of an HPLC (high-performance liquid chromatography).
  • This HPLC purification was conducted by using a column (SISEIDO Capsule-Pack C-18 SG120, diameter: 46 mm, length: 250 mm) in a manner such that a gradient was applied to the mobile phase at a flow rate of 12 ml/min by use of a solution comprising 0.1 % of trifluoroacetic acid (TFA) and 5 % of acetonitrile (CH 3 CN) in pure water; and a solution comprising 0.1 % of TFA and 50 % of acetonitrile (CH 3 CN) in pure water.
  • TFA trifluoroacetic acid
  • CH 3 CN acetonitrile
  • the resultant peptide solution was poured into wells of a 96-well micro-plate (trade name: ELISA PLATE 68667, mfd. by NUNC Co.) so that 100 ⁇ l of the solution was poured into each of the wells. Then, the micro-plate was left standing at 37 °C for 60 minutes, thereby to solid phase-fix the above antigenic peptide to the micro-plate.
  • the resultant plate coated with the gelatine was dried at 25 °C for six hours, and thereafter, was stored at 4 °C until the plate was used for analyzing specimens.
  • HCV-RNA unit: mEQ/ml
  • HCV-RNA measuring reagent trade name: Quantiplex HCV-RNA, mfd. by Chiron Co.
  • the thus diluted specimen was poured into each of the micro-plate wells in an amount of 100 ⁇ l per one well, and then was allowed to react with the above antigenic peptide at 37 °C for 60 minutes (reaction between antigenic peptide - HCV antibody).
  • OPD o-phenylene diamine
  • a micro-plate to which an antigenic peptide ckk-c22 had been solid-phase fixed was prepared in the same manner as in Example 3 except for using the antigenic peptide ckk-c22 obtained in Example 2 instead of the antigenic peptide ckk-c12 used in Example 3.
  • Specimens (20 kinds), the HCV genotypes of which had preliminarily been determined, were analyzed in the same manner as in Example 4 except for using the thus obtained micro-plate.
  • the serotypes of these specimens were determined on the basis of the thus obtained measured absorbance data and those obtained in the above Example 4.
  • the absorbance obtained by using the antigenic peptide ckk-c12 i.e., a peptide corresponding to serotype-1
  • the absorbance obtained by using the antigenic peptide ckk-c22 i.e., a peptide corresponding to serotype-2 were compared with each other.
  • the serotype was judged to be a serotype corresponding to the higher absorbance value (i.e., when the higher one is ckk-c12 side, it was judged to be serotype-1; and when the higher one is ckk-c22 side, it was judged to be serotype-2).
  • the symbol “1” denotes “type-1”
  • the symbol “2” denotes “type-2”
  • the symbol “0” denotes “negative” (determination was impossible)
  • genotype V also includes such cases, the genotype determinations of which were impossible, or indistinguishable (inclusive of "mixed types") by use of the PCR method.
  • a micro-plate to which an antigenic peptide had been solid-phase fixed was prepared in the same manner as in Example 3 except for using the antigenic peptide ckk-n1 or ckk-n2 corresponding to the NS-4 region obtained in Example 2, respectively, instead of the antigenic peptide ckk-c12 used in Example 3.
  • the absorbance (ckk-n1) and the absorbance (ckk-n2) were compared with each other.
  • the serotype was judged to be a serotype corresponding to the higher absorbance value (i.e., when the higher one is ckk-n1 side, it was judged to be serotype-1; and when the higher one is ckk-n2 side, it was judged to be serotype-2).
  • the measurement of the absorbance and the determination of the serotype were conducted in the same manner as in Example 6 except for changing the dilution factor for the specimen to 500 times.
  • a micro-plate to which an antigenic peptide had been solid-phase fixed was prepared in the same manner as in Example 3 except for using the antigenic peptide ckk-n3 or ckk-n4 corresponding to the NS-5 region obtained in Example 2, respectively, instead of the antigenic peptide ckk-c12 used in Example 3.
  • the absorbance (ckk-n3) and the absorbance (ckk-n4) were compared with each other.
  • the serotype was judged to be a serotype corresponding to the higher absorbance value (i.e., when the higher one is ckk-n3 side, it was judged to be serotype-1; and when the higher one is ckk-n4 side, it was judged to be serotype-2).
  • a micro-plate to which an antigenic peptide had been solid-phase fixed was prepared in the same manner as in Example 3 except for using the antigenic peptide ckk-c1 or ckk-c2 (each of which was a peptide comprising 20 amino acids) corresponding to the core region obtained in Example 2, respectively, instead of the antigenic peptide ckk-c12 used in Example 3.
  • the absorbance (ckk-c1)and the absorbance (ckk-c2) were compared with each other.
  • the serotype was judged to be a serotype corresponding to the higher absorbance value (i.e., when the higher one is ckk-c1 side, it was judged to be serotype-1; and when the higher one is ckk-c2 side, it was judged to be serotype-2).
  • genotype-I and genotype-II showed a good correlation with the serotype-I.
  • genotype-III and genotype-IV showed a good correlation with the serotype-II.
  • the present invention provides an antigenic peptide having an amino acid sequence peculiar to the HCV, and a method of measuring an HCV antibody by using such a peptide.
  • the present invention it is possible to enhance the sensitivity to the core region so as to judge the serotype of a specimen simply and accurately, while suppressing a cross reaction or non-specific reaction. Therefore, according to the present invention, for example, it is possible to preliminarily predict the effect of interferon treatment on the basis of the simple and accurate serotype determination.
  • the present invention it is possible to simply observe the course of the curing or treatment for hepatitis C, on the basis of the measurement of the HCV antibody titer according to the present invention. More specifically, according to the present invention, it is also possible to utilize the serotype from an epidemiological viewpoint, and to obtain a knowledge on the past infection (antibody titer with respect to the core region) and on the current hepatitis C (antibody titer with respect to the NS-4 and/or NS-5 region).
  • the present invention provides an antigen for measuring an HCV antibody, which enables simple determination of the HCV antibody wherein the HCV genotype-I and genotype-II can be distinguished from the HCV genotype-III and genotype-IV.
  • the present invention provides an antigen for measuring an HCV antibody, which enables the determination of the HCV antibody wherein the HCV genotype-I and genotype-II can be distinguished from the HCV genotype-III and genotype-IV; and further a cross-reaction or non-specific reaction is suppressed.
  • the present invention provides a method of simply measuring an HCV antibody, wherein the HCV genotype-I and genotype-II can be distinguished from the HCV genotype-III and genotype-IV.
  • the present invention provides a method of simply measuring an HCV antibody, wherein the HCV genotype-I and genotype-II can be distinguished from the HCV genotype-III and genotype-IV; and further a cross-reaction or non-specific reaction is suppressed.
  • the present invention provides a method of measuring an HCV antibody at a low cost, wherein the HCV genotype-I and genotype-II can be distinguished from the HCV genotype-III and genotype-IV.

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EP95917489A 1995-04-28 1995-04-28 Antigene peptidsubstanz und immotestverfahren Withdrawn EP0829488A4 (de)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP1995/000849 WO1996034013A1 (fr) 1995-04-28 1995-04-28 Compose peptidique antigenique et methode de dosage immunologique

Publications (2)

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EP0829488A1 true EP0829488A1 (de) 1998-03-18
EP0829488A4 EP0829488A4 (de) 1999-04-07

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EP (1) EP0829488A4 (de)
KR (1) KR19990008122A (de)
AU (1) AU2352995A (de)
WO (1) WO1996034013A1 (de)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015516A2 (en) * 1990-04-06 1991-10-17 Genelabs Incorporated Hepatitis c virus epitopes
WO1992010514A2 (en) * 1990-12-14 1992-06-25 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis c virus
EP0516859A1 (de) * 1990-11-29 1992-12-09 Toray Industries, Inc. Non-a non-b hepatitisvirus-antigenprotein
WO1993017111A1 (en) * 1992-02-28 1993-09-02 The Wellcome Foundation Limited Hepatitis c virus peptides
EP0593290A2 (de) * 1992-10-16 1994-04-20 Evernew Biotech Inc. Kernantigenprotein des Hepatitis C-Virus, sowie diagnostisches Verfahren und Satz von Reagenzen

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UA40572C2 (uk) * 1991-06-24 2001-08-15 Чірон Корпорейшн Поліпептид, що містить укорочену послідовність вірусу гепатиту с, ізольований епітоп, реагент для імуноаналізу на вірус гепатиту с (варіанти), спосіб виявлення наявності антитіл (варіанти)
DE4209215A1 (de) * 1991-07-04 1993-01-07 Boehringer Mannheim Gmbh Hcv peptidantigene und verfahren zur bestimmung von hcv
AU2683792A (en) * 1991-09-16 1993-04-27 Genelabs Technologies, Inc. Peptide based hepatitis c virus immunoassays
NZ299048A (en) * 1992-03-06 1998-09-24 Innogenetics Sa Nv Modified peptides corresponding to immunologically useful epitopes in hiv, hcv and htlv and their use especially when biotinylated
UA46707C2 (uk) * 1993-05-10 2002-06-17 Чірон Корпорейшн Спосіб типування вірусу гепатиту с (варіанти), набір поліпептидів (варіанти), реагент, що включає комбінацію поліпептидів, які містять типоспецифічні епітопи гепатиту с (варіанти)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015516A2 (en) * 1990-04-06 1991-10-17 Genelabs Incorporated Hepatitis c virus epitopes
EP0516859A1 (de) * 1990-11-29 1992-12-09 Toray Industries, Inc. Non-a non-b hepatitisvirus-antigenprotein
WO1992010514A2 (en) * 1990-12-14 1992-06-25 Innogenetics N.V. Synthetic antigens for the detection of antibodies to hepatitis c virus
WO1993017111A1 (en) * 1992-02-28 1993-09-02 The Wellcome Foundation Limited Hepatitis c virus peptides
EP0593290A2 (de) * 1992-10-16 1994-04-20 Evernew Biotech Inc. Kernantigenprotein des Hepatitis C-Virus, sowie diagnostisches Verfahren und Satz von Reagenzen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9634013A1 *

Also Published As

Publication number Publication date
EP0829488A4 (de) 1999-04-07
AU2352995A (en) 1996-11-18
WO1996034013A1 (fr) 1996-10-31
KR19990008122A (ko) 1999-01-25

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